Duplexed parvovirus vectors

a parvovirus and vector technology, applied in the field of parvovirus-based gene delivery vectors, to achieve the effects of improving the efficiency of transduction, rapid onset, and increasing the level of transgene expression

Inactive Publication Date: 2008-12-16
THE UNIV OF NORTH CAROLINA AT CHAPEL HILL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0008]The single-stranded nature of the AAV genome may impact the expression of rAAV vectors more than any other biological feature. Rather than rely on potentially variable cellular mechanisms to provide a complementary-strand for rAAV vectors, it has now been found that this problem may be circumvented by packaging both strands as a single DNA molecule. In the studies described herein, an increased efficiency of transduction from duplexed vectors over conventional rAAV was observed in HeLa cells (5-140 fold). More importantly, unlike conventional single-stranded AAV vectors, inhibitors of DNA replication did not affect transduction from the duplexed vectors of the invention. In addition, the inventive duplexed parvovirus vectors displayed a more rapid onset and a higher level of transgene expression than did rAAV vectors in mouse hepatocytes in vivo. All of these biological attributes support the generation and characterization of a new class of parvovirus vectors (delivering duplex DNA) that significantly contribute to the ongoing development of parvovirus-based gene delivery systems.

Problems solved by technology

Rather than rely on potentially variable cellular mechanisms to provide a complementary-strand for rAAV vectors, it has now been found that this problem may be circumvented by packaging both strands as a single DNA molecule.

Method used

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  • Duplexed parvovirus vectors
  • Duplexed parvovirus vectors
  • Duplexed parvovirus vectors

Examples

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example 1

Materials and Methods

[0170]Plasmids. The rAAV plasmids expressing green fluorescent protein (GFP) were constructed from the previously described pTRBSUF-2 (a gift from Nick Muzyczka). First, the humanized GFP coding sequence was replaced with the enhanced GFP (eGFP) (Clonetech) to create the plasmid, pTR-CMV-GFPneo. This plasmid generated the rAAV-GFPneo vector. Second, the Sal I fragment containing the neo coding region and SV40 promoter was deleted to create pTR-CMV-GFP. The vector from this plasmid was referred to as rAAV-GFP in this report.

[0171]The plasmid, p43mEpo, a gift from Barry Byrne, contained the mouse erythropoietin gene under the control of the CMV promoter and generated a rAAV replicon (rAAVmEpo) of less than half the wtAAV length. A longer version of this construct (pmEpo-λ) was made by inserting the 2.3 kb Hind III fragment from λ phage into a Cla I site between the polyadenylation signal and the downstream AAV terminal repeat. The rAAV-LacZ vector was generated fr...

example 2

Generation of Duplexed Vectors

[0176]A rAAV plasmid construct (pTR-CMV-GFP), with a replicon size of 2299 nucleotides, was used to generate a viral vector stock (rAAV-GFP) by conventional methods. The predicted size of the dimeric replicative form of this vector was 4474 nucleotides (FIG. 1), which was 95.6% of the wt AAV genome length. The viral vectors were fractionated by isopycnic gradient centrifugation in CsCl and the vDNA content of each fraction was analyzed on alkaline agarose gels (FIG. 2). Phospholmager scans were used to quantify the vDNA specific bands from each fraction. Under denaturing conditions, the self-complementary dimer DNA (FIG. 2, panel a, fractions 10-13) ran at approximately twice the length of the monomeric genome. The hybridizing material in fractions 2-4 is unpackaged replicative form DNA that sediments at the bottom of the gradient. Although a DNase step was included in the vector purification (see methods), the treatment was not intended to be exhaustiv...

example 3

Transduction with Duplexed versus Monomeric Vectors and Effects of Ad co-Infection

[0178]The transducing efficiency of the scAAV-GFP (FIG. 2, panel a, fraction 11) was compared with the homologous monomer (fraction 13), as well as the GFPneo and LacZ vectors (FIG. 2, panels b and c, fractions 13 and 12, respectively) in HeLa cells infected at low multiplicity (FIG. 3). The particle numbers were calculated from the specific, full-length vDNA Phospholmager signals in each fraction on the Southern blot, after correction for monomeric versus dimeric DNA copy number. Thus, each duplexed virus contains two copies of the transgene as a single molecule, in the inverted repeat orientation, while each monomeric particle contains one single-stranded copy.

[0179]The scAAV-GFP vector (fraction 11), containing approximately 90% dimer virus, yielded a 5.9:1 ratio of physical particles to transducing units, thus bearing out the prediction of high transducing efficiency. Fraction 13 from the same grad...

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Abstract

The present invention provides duplexed parvovirus vector genomes that are capable under appropriate conditions of forming a double-stranded molecule by intrastrand base-pairing. Also provided are duplexed parvovirus particles comprising the vector genome. Further disclosed are templates and methods for producing the duplexed vector genomes and duplexed parvovirus particles of the invention. Methods of administering these reagents to a cell or subject are also described. Preferably, the parvovirus capsid is an AAV capsid. It is further preferred that the vector genome comprises AAV terminal repeat sequences.

Description

RELATED APPLICATION INFORMATION[0001]This application claims priority under 35 U.S.C. § 371 from PCT Application No. PCT / US01 / 17587 (published under PCT Article 21(2) in English), filed on May 31, 2001, which claims the benefit of U.S. Provisional Application No. 60 / 208,604, filed on Jun. 1, 2000, the disclosures of which are incorporated by reference herein in their entireties.STATEMENT OF FEDERAL SUPPORT[0002]The present invention was made, in part, with the support of grant numbers HL51818, HL 48347, and DK 54419 from the National Institutes of Health. The United States government has certain rights to this invention.FIELD OF THE INVENTION[0003]The present invention relates to reagents for gene delivery. More particularly, the present invention relates to improved parvovirus-based gene delivery vectors.BACKGROUND OF THE INVENTION[0004]Adeno-associated virus (AAV) is a nonpathogenic, helper dependent member of the parvovirus family. One of the identifying characteristics of this g...

Claims

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Application Information

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Patent Type & Authority Patents(United States)
IPC IPC(8): C12N15/86A61K48/00C12N15/00C12N15/64C12N15/09A61K35/76A61K38/00A61K38/44A61K39/395A61P35/00C12N5/10C12N7/00C12N7/02C12N15/864C12Q1/70
CPCC12N7/00C12N15/864C12N15/86A61K48/00C12N2750/14121C12N2750/14143A61P31/00A61P31/12A61P33/00A61P35/00A61P37/00
Inventor SAMULSKI, RICHARD JUDEMCCARTY, DOUGLAS M.
Owner THE UNIV OF NORTH CAROLINA AT CHAPEL HILL
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